WO2017100674A1 - Procédé de purification évolutif pour virus adéno-associé (aav) 1 - Google Patents

Procédé de purification évolutif pour virus adéno-associé (aav) 1 Download PDF

Info

Publication number
WO2017100674A1
WO2017100674A1 PCT/US2016/065974 US2016065974W WO2017100674A1 WO 2017100674 A1 WO2017100674 A1 WO 2017100674A1 US 2016065974 W US2016065974 W US 2016065974W WO 2017100674 A1 WO2017100674 A1 WO 2017100674A1
Authority
WO
WIPO (PCT)
Prior art keywords
buffer
aavl
nacl
aav
intermediates
Prior art date
Application number
PCT/US2016/065974
Other languages
English (en)
Inventor
Martin Lock
Mauricio Alvira
Original Assignee
The Trustees Of The University Of Pennsylvania
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by The Trustees Of The University Of Pennsylvania filed Critical The Trustees Of The University Of Pennsylvania
Priority to US16/060,406 priority Critical patent/US11015173B2/en
Publication of WO2017100674A1 publication Critical patent/WO2017100674A1/fr
Priority to US17/328,687 priority patent/US11713450B2/en
Priority to US18/333,718 priority patent/US20230323312A1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C12N7/02—Recovery or purification
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
    • B01D15/16—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to the conditioning of the fluid carrier
    • B01D15/166—Fluid composition conditioning, e.g. gradient
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/36—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving ionic interaction
    • B01D15/361—Ion-exchange
    • B01D15/363—Anion-exchange
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 - B01D15/36
    • B01D15/3804—Affinity chromatography
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J41/00—Anion exchange; Use of material as anion exchangers; Treatment of material for improving the anion exchange properties
    • B01J41/04—Processes using organic exchangers
    • B01J41/05—Processes using organic exchangers in the strongly basic form
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J41/00—Anion exchange; Use of material as anion exchangers; Treatment of material for improving the anion exchange properties
    • B01J41/08—Use of material as anion exchangers; Treatment of material for improving the anion exchange properties
    • B01J41/12—Macromolecular compounds
    • B01J41/13—Macromolecular compounds obtained otherwise than by reactions only involving unsaturated carbon-to-carbon bonds
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J41/00—Anion exchange; Use of material as anion exchangers; Treatment of material for improving the anion exchange properties
    • B01J41/20—Anion exchangers for chromatographic processes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J47/00—Ion-exchange processes in general; Apparatus therefor
    • B01J47/02—Column or bed processes
    • B01J47/022—Column or bed processes characterised by the construction of the column or container
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C12N15/864—Parvoviral vectors, e.g. parvovirus, densovirus
    • C12N15/8645—Adeno-associated virus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
    • G01N21/33—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using ultraviolet light
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14151—Methods of production or purification of viral material

Definitions

  • This invention describes a novel scalable method for producing rAAV suitable for clinical applications.
  • rAAV recombinant adeno-associated viruses
  • Adeno-associated virus a member of the Parvovirus family, is a small, non-enveloped virus.
  • AAV particles comprise an AAV capsid composed of 60 capsid protein subunits, VPl, VP2 and VP3, which enclose a single-stranded DNA genome of about 4.7 kilobases (kb). These VPl, VP2 and VP3 proteins are present in a predicted ratio of about 1 : 1 : 10, and are arranged in an icosahedral symmetry.
  • AAV is assigned to the genus, Dependoviru , because the virus was discovered as a contaminant in purified adenovirus stocks.
  • AAV's life cycle includes a latent phase and an infectious phase. Replication occurs by conversion of the linear single stranded DNA genome to a duplex form, and subsequent amplification, from which progeny single strands are rescued, replicated, and packaged into capsids in the presence of helper functions.
  • the properties of non-pathogenicity, broad host range of infectivity, including non-dividing cells, and integration make AAV an attractive delivery vehicle.
  • Recombinant AAV particles are produced in permissive (packaging) host cell cultures and co-expression of helper virus AAV rep and AAV cap genes are required, for replication and packaging, the recombinant genome into the viral particle.
  • Genes necessary for genome replication, capsid formation and genome packaging can be expressed from transfected plasmids, integrated into the host cell genome or introduced to the cell by recombinant viruses.
  • cells are lysed to release rAAV particles and maximize yield of recovered rAAV.
  • the cell lysate contains various cellular components such as host cell DNA, host cell proteins, media components, and in some instances, helper virus or helper virus plasmid DNA, which must be separated from the rAAV vector before it is suitable for in vivo use.
  • rAAV production include the use of non-adherent cell suspension processes in stirred tank bioreactors and production conditions whereby rAAV vectors are released into the media or supernatant reducing the concentration of host cellular components present in the production material but still containing appreciable amounts of in-process impurities. See US Patent No. 6,566,118 and PCT WO
  • rAAV particles may be collected from the media and/or cell lysate and further purified.
  • Certain previously described purification methods for rAAV are not scalable and/or not adaptable to good manufacturing practices, including, e.g., cesium chloride gradient centrifugation and iodixanol gradient separation. See, e.g., M. Potter et al, Molecular Therapy - Methods & Clinical Development (2014), 1 : 14034, pp 1-8.
  • This affinity capture method focuses on purifying rAAV vectors from in-process production components of the cell culture including helper virus, as well as helper virus proteins, cellular proteins, host cell DNA, and media components present in the rAAV production stock.
  • the affinity capture method described for purifying rAAV 1, 2, 3 and 5 particles is designed to purify rAAV from host cell and helper virus contaminants, but not to separate AAV particles from empty AAV capsids lacking packaged genomic sequences.
  • the present invention provides a scalable method for efficiently separating genome-containing AAVl vector particles (full) from genome-deficient rAAVl intermediates (empty capsids). Also provided are purified AAVl vector particles.
  • AAVl intermediates comprises subjecting a mixture comprising recombinant AAVl viral particles and AAVl vector intermediates/byproducts to fast performance liquid chromatography (FPLC), wherein the AAVl viral particles and AAVl intermediates are bound to a strong anion exchange resin equilibrated at a pH of about 9.8 and subjected to a salt gradient while monitoring the eluate for ultraviolet absorbance at about 260nm and about 280nm.
  • the AAVl full capsids are collected from a fraction which is eluted when the ratio of A260/A280 reaches an inflection point.
  • the full capsids are collected from the eluted fraction(s) characterized by having a higher peak (area under the curve) at an absorbance of 260nm as compared to the peak (area under the curve) at an absorbance of 280 nm.
  • the majority of the fractions observed for the process of the invention have a higher amount of empty capsids (higher peak/area under curve at A280).
  • the absorbance peak at 260 nm being equal to or exceeding the absorbance peak at 280 nm is indicative of the fraction containing the full capsids.
  • FPLC chromatography
  • a scalable method for separating full AAVl viral particles from AAVl intermediates by using an anti-AAV antibody based affinity capture resin followed by an anion exchange resin.
  • the mixture containing the AAVl viral particles and AAVl intermediates is loaded onto the affinity resin in a buffer having a high salt concentrations, e.g., about 400 nM NaCl to about 650 mM NaCl or another salt(s) having an equivalent ionic strength.
  • the wash step for the affinity resin is thereafter performed at an even higher salt concentration, e.g., in the range of about 750 mM to about 1 M NacCl or equivalent.
  • the AAVl mixture is maintained at a salt concentration of about 400 mM NaCl to about 650 mM NaCl, or equivalent prior to being applied to the anion exchange resin column.
  • the rAAVl mixture is maintained at this salt concentration following concentration and prior to loading onto the affinity resin.
  • a method for separating AAVl viral particles from AAVl capsid intermediates comprising: (a) mixing a suspension comprising recombinant AAVl viral particles and AAV 1 vector intermediates and a Buffer A comprising 20mM to 50 mM Bis-Tris propane (BTP) and a pH of about 9.8; (b) loading the suspension of (a) onto a strong anion exchange resin column; (c) washing the loaded anion exchange resin with Buffer 1% B which comprises a salt having the ionic strength of lOmM to 40 mM NaCl and BTP with a pH of about 9.8; (d) applying an increasing salt concentration gradient to the loaded and washed anion exchange resin, wherein the salt gradient is the equivalent of 10 mM to about 40 mM NaCl; and (e) collecting rAAVl particles from the eluate, where the rAAVl particles are at least about 90% purified from AAVl intermediates.
  • a scalable method is provided for separating
  • pharmacologically active recombinant AAVl viral particles containing DNA genomic sequences from inert genome-deficient (empty) AAVl vector intermediates comprising: (a) forming a loading suspension comprising: recombinant AAVl viral particles and empty AAV 1 capsid which have been purified to remove contaminants from an AAV producer cell culture in which the particles and intermediates were generated; and a Buffer A comprising 20 mM Bis-Tris propane (BTP) and a pH of about 9.8; (b) loading the suspension of (a) onto a strong anion exchange resin, said resin being in a vessel having an inlet for flow of a suspension and/or solution and an outlet permitting flow of eluate from the vessel; (c) washing the loaded anion exchange resin with Buffer 1% B which comprises lOmM NaCl and 20mM BTP with a pH of about 9.8; (d) applying an increasing salt concentration gradient to the loaded and washed anion exchange resin, wherein the salt gradient ranges from 10
  • the affinity resin separation comprises: (i) equilibrating the affinity resin with Buffer Al which comprises about 200 mM to about 600 mM NaCl, about 20 mM Tris-Cl and a neutral pH prior to applying the material to the affinity resin; (ii) washing the loaded resin of (a) with Buffer CI which comprises about 800 mM NaCl to about 1200 mM NaCl, 20 mM Tris-Cl and a neutral pH; (iii) washing the Buffer CI -washed resin of (b) with Buffer Al to reduce salt concentration; (iv) washing the affinity resin of (c) with Buffer B which comprises about 200 nM to about 600 nM NaCl, 20 mM Sodium Citrate, pH about 2.4 to about 3; and (v) collecting the eluate of (iv) which comprises the full AAV1 particles and the empty AAV1 capsid fraction for loading onto the anion exchange resin.
  • Buffer Al which comprises about 200 mM to about 600
  • vector preparations are provided that have less than 5% contamination with AAV intermediates (including AAV empty capsids). In another aspect, vector preparations are provided that have less than 2% contamination with AAV empty capsids, or less than 1% contamination with AAV empty capsids. In a further aspect, AAV compositions are provided which are substantially free of AAV empty capsids.
  • FIG 1 shows a chromatogram of AVB affinity -purified AAV1 vector preparation (1.2x 10 1 GC) with a single -stranded genome run on a O. lmL CIMac QA column.
  • the run was performed with 20mM Bis-Tris-Propane (BTP) pH9.5 as the loading buffer (buffer A) and 20mM BTP pH9.5-lM NaCl as the column strip buffer (Buffer B).
  • Buffer A loading buffer
  • Buffer B 20mM BTP pH9.5-lM NaCl
  • Buffer B 20mM Bis-Tris-Propane
  • Buffer B 20mM BTP pH9.5-lM NaCl
  • A260 line extending highest at peak PI
  • A280 line extending second highest at peak PI
  • programmed conductivity line extending from Y axis and connecting at -11%)
  • actual conductivity line extending from Y axis at -7%) profiles are shown.
  • Percentages of the highest peak of Absorbance (mAU) are shown on the y axis.
  • Run volume (mL) is shown as solid line beneath the x axis while buffer is indicated on the x axis above the run volume.
  • the major peaks (labeled PI - P3) are indicated.
  • the peak fraction pool boundaries are indicated by vertical dotted lines on the x axis and the percent recovery of loaded vector genomes in each peak fraction pool are indicated below the axis.
  • FIG 2 shows a chromatogram of AVB affinity -purified AAV1 vector preparation (1.8x 10 1 GC) run on a 0. ImL CIMac QA column.
  • the run was performed with 20mM Bis-Tris-Propane (BTP) pHlO.O as the loading buffer (buffer A) and 20mM BTP pH10.0-lM NaCl as the column strip buffer (Buffer B).
  • BTP Bis-Tris-Propane
  • Buffer A 20mM BTP pH10.0-lM NaCl
  • Buffer B Buffer B
  • a 60 CV linear salt gradient from 1- 19% Buffer B was used to elute vector and the column was stripped with 100% Buffer B.
  • the flow rate was maintained at 2mL/min throughout the run.
  • A260 line extending highest at peak P2
  • A280 line extending second highest at peak P2 and highest at peak P3
  • programmed conductivity line extending from Y axis and connecting at -40 mAU
  • actual conductivity line extending from Y axis and connecting at -15 mAU
  • Absorbance mAU
  • Run volume mL
  • buffer is indicated on the x axis above the run volume.
  • the major peaks (labeled PI - P3) are indicated.
  • the peak fraction pool boundaries are indicated by vertical dotted lines on the x axis and the percent recovery of loaded vector genomes in each peak fraction pool are indicated below the axis.
  • FIG 3 shows a chromatogram of an AVB affinity-purified AAV1 vector preparation (2.64x10 14 GC) run on an 8mL CIMmultus QA column and the chromatogram is shown.
  • the run was performed with 20mM Bis-Tris-Propane (BTP) pH9.8 as the loading buffer (buffer A) and 20mM BTP pH9.8-lM NaCl as the column strip buffer (Buffer B).
  • BTP Bis-Tris-Propane
  • Buffer B 20mM BTP pH9.8-lM NaCl
  • Buffer B Buffer B
  • a 60 CV linear salt gradient from 1- 19% Buffer B was used to elute vector and the column was stripped with 100% Buffer B.
  • the column was loaded at a flow rate of lOmL/min and eluted at 20mL/min.
  • A260 line extending highest at peak PI
  • A280 line extending second highest at peak PI and highest at peak P2
  • programmed conductivity line extending
  • a scalable technology for production of purified rAAVl for use in a variety of gene transfer and/or other applications is provided.
  • the method purifies rAAVl viral particles from production culture contaminants such as helper virus, helper virus proteins, plasmids, cellular proteins and DNA, media components, serum proteins, AAV rep proteins, unassembled AAV VP1, AAVV P2 and AAV VP3 proteins, and the like.
  • the method provided herein is particularly well suited for separating full rAAVl viral particles from rAAV intermediates.
  • the method is useful for certain other AAV which share certain required structural with AAV1 as described herein.
  • the method for separating full AAV1 viral particles from empty AAV1 intermediates comprises subjecting a mixture comprising recombinant AAV1 viral particles and AAV 1 vector intermediates to fast performance liquid
  • AAV1 viral particles and AAV1 intermediates are bound to a strong anion exchange resin equilibrated at a pH of about 9.8 and subjected to a salt gradient while monitoring eluate for ultraviolet absorbance at about 260 nm and about 280 nm, respectively.
  • AAV1 capsids having genomic sequences packaged therein (“full") and 260/280 absorbance ratios less than 1 is characteristic of AAV1 intermediates as defined herein.
  • the production cell culture may yield a mixture of rAAVl “full” and rAAVl “empty” or other intermediates in which 50% or greater are intermediates (including empties), at least 60% are intermediates, or greater than 70% are intermediates.
  • more or less of the genome copies are "empty”; as a consequence, a corresponding amount of eluted fractions are characterized by having 280 nm peaks (and corresponding larger areas under the curve which are larger than 260nm peaks).
  • Fractions characterized by peaks (and corresponding larger areas under the curve at an absorbence of about 260 nm (A260) that are higher than the corresponding peaks at 260 nm (A260/280 ratio is >1 ) are highly enriched in full rAAVl particles.
  • the AAV1 full capsids are collected from a fraction which is eluted when the peak for A260 crosses over and exceeds the peak for A280 (i.e., reaches an inflection point).
  • recombinant AAV1 viral particle refers to nuclease-resistant particle (NRP) which has an AAV1 capsid, the capsid having packaged therein a heterologous nucleic acid molecule comprising an expression cassette for a desired gene product.
  • NRP nuclease-resistant particle
  • Such an expression cassette typically contains an AAV 5' and/or 3' inverted terminal repeat sequence flanking a gene sequence, in which the gene sequence is operably linked to expression control sequences.
  • transgene genomic sequences This may also be referred to as a "full" AAV capsid.
  • Such a rAAV viral particle is termed "pharmacologically active" when it delivers the transgene to a host cell which is capable of expressing the desired gene product carried by the expression cassette.
  • rAAV particles are referred to as DNase resistant (DRP).
  • DNase DNase resistant
  • exonucleases may also be used in the purification steps described herein, to remove contaminating nucleic acids.
  • Such nucleases may be selected to degrade single stranded DNA and/or double-stranded DNA, and RNA.
  • Such steps may contain a single nuclease, or mixtures of nucleases directed to different targets, and may be endonucleases or exonucleases.
  • nuclease-resistant indicates that the AAV capsid has fully assembled around the expression cassette which is designed to deliver a transgene to a host cell and protects these packaged genomic sequences from degradation (digestion) during nuclease incubation steps designed to remove contaminating nucleic acids which may be present from the production process.
  • AAV1 capsid refers to the AAV capsid having the amino acid sequence of GenBank, accession: NP 049542 (identical to GenBank
  • AAD27757 which is incorporated by reference herein and reproduced in SEQ ID NO: 1.
  • the methods provided herein can be used to purify other AAV having a capsid highly related to the AAV1 capsid. For example, AAV having sequences having about 99% identity to the referenced amino acid sequence in
  • NP_049542 and US Patent Nos. 6759237, 7105345, 7186552 may be purified using the methods described herein, provided that the integrity of the ligand-binding site for the affinity capture purification is maintained and the change in sequences does not substantially alter the pH range for the capsid for the ion exchange resin purification.
  • Methods of generating the capsid, coding sequences therefore, and methods for production of rAAV viral vectors have been described. See, e.g., Gao, et al, Proc. Natl. Acad. Sci. U.S.A. 100 (10), 6081-6086 (2003), US Patent Nos. 6759237, 7105345, 7186552 and US Patent Publication 2013/0045186A1.
  • identity or “percent sequence identity” may be readily determined for amino acid sequences, over the full-length of a protein, a subunit, or a fragment thereof.
  • a fragment is at least about 8 amino acids in length, and may be up to about 700 amino acids. Examples of suitable fragments are described herein.
  • identity is determined in reference to “aligned” sequences. "Aligned" sequences or
  • Alignments refer to multiple nucleic acid sequences or protein (amino acids) sequences, often containing corrections for missing or additional bases or amino acids as compared to a reference sequence. Alignments are performed using a variety of publicly or commercially available Multiple Sequence Alignment Programs.
  • Such programs include, “Clustal W”, “CAP Sequence Assembly”, “MAP”, and “MEME”, which are accessible through Web Servers on the internet. Other sources for such programs are known to those of skill in the art. Alternatively, Vector NTI utilities are also used. There are also a number of algorithms known in the art that can be used to measure nucleotide sequence identity, including those contained in the programs described above. As another example, polynucleotide sequences can be compared using FastaTM, a program in GCG Version 6.1. FastaTM provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences.
  • percent sequence identity between nucleic acid sequences can be determined using FastaTM with its default parameters (a word size of 6 and the NOP AM factor for the scoring matrix) as provided in GCG Version 6.1, herein incorporated by reference.
  • Multiple sequence alignment programs are also available for amino acid sequences, e.g., the "Clustal X”, “MAP”, “PIMA”, “MSA”, “BLOCKMAKER”, “MEME”, and “Match-Box” programs. Generally, any of these programs are used at default settings, although one of skill in the art can alter these settings as needed.
  • one of skill in the art can utilize another algorithm or computer program which provides at least the level of identity or alignment as that provided by the referenced algorithms and programs. See, e.g., J. D. Thomson et al, Nucl. Acids Res., "A comprehensive comparison of multiple sequence alignments", 27(13):2682-2690 (1999).
  • AAVl intermediate or "AAVl vector intermediate” refers to an assembled rAAV capsid which lacks genomic sequences packaged therein. These may also be termed an "empty" capsid. Such a capsid may contain no detectable genomic sequences of an expression cassette, or only partially packaged genomic sequences which are insufficient to achieve expression of the gene product. These empty capsids are non-functional to transfer the gene of interest to a host cell.
  • a method for separating rAAVl particles having packaged genomic sequences from genome-deficient AAVl intermediates involves subjecting a suspension comprising recombinant AAVl viral particles and AAVl capsid intermediates to fast performance liquid chromatography, wherein the AAVl viral particles and AAVl intermediates are bound to a strong anion exchange resin equilibrated at a pH of about 9.8 and subjected to a salt gradient while monitoring eluate for ultraviolet absorbance at about 260 and about 280.
  • the pH may be as low as 9.6 or as high as 10.0.
  • the AAVl full capsids are collected from a fraction which is eluted when the ratio of A260/A280 reaches an inflection point.
  • FPLC Fast protein liquid chromatography
  • the mobile phase is an aqueous solution, or "buffer”.
  • the buffer flow rate may be controlled by gravity or a pump (e.g., a positive-displacement pump) and can be kept constant or varied.
  • the composition of the buffer can be varied by drawing fluids in different proportions from two or more external reservoirs.
  • the stationary phase described herein is a strong anion exchange resin, typically composed of beads. These beads may be packed into a vessel, e.g., a cylindrical glass or plastic column, or another suitable vessel.
  • volumes of the mobile phase are described as "column volumes”. These volumes may be extrapolated to other vessel shapes and designs.
  • the eluate from the anion exchange resin column or other vessel is monitored for ultraviolet absorbance at about 260 nm and 280 nm.
  • "full" AAV1 capsids are characterized by having a UV absorbance of about 260 nm
  • empty capsids are characterized by having a UV absorbance of about 280 nm.
  • the majority of the eluate fractions contain empty capsids and as the salt gradient progresses, the majority of the eluate is characterized by a curve for A280 exceeding that of A260.
  • A260/A280 greater than 1) one can selectively collect the "full capsids" until such time as the ratio reverts to A280/A260 greater than 1.
  • this fraction(s) selectively collected at the inversion point is characterized by having the total collected rAAV contain at least about 90% "full capsids", and preferably, at least 95% "full capsids". In a further embodiment, these fractions may be characterized by having a ratio of "intermediate" to "full” less than 0.75, more preferably 0.5, preferably less than 0.3.
  • an "anion exchange resin” refers to an insoluble matrix or solid support (e.g., beads) capable of having a surface ionization over a pH range of about 1 to about 14.
  • a strong anion exchange resin is a solid support having a surface coated with quatemized polyethyleneimine.
  • An example of such a strong anionic exchange resin is the solid support of the CIMultus QATM column.
  • the anion exchange resin may be a quaternary amine ion exchange resin.
  • the anion exchange resin comprises trimethylamine and a support matrix comprising poly(glycidyl methacrylate - co- ethylene dimethacrylate).
  • other suitable anion exchange resins may be selected.
  • An example of such a strong anionic exchange resin is that of the POROS HQTM column. The resins for the columns listed above can be obtained from
  • the anion exchange material may be in the form of a monolith column or a traditional bead-based column.
  • the ion exchange material can be in a column having a capacity of 0 to 0.5 mL column, 1 mL column, and more preferably, at least an 8 mL column, a 10 mL column, a 20 mL column, a 30 mL column, a 50 mL column, a 100 mL column, a 200 mL column, a 300 mL column, a 400 mL column, a 500 mL column, a 600 mL column, a 700 mL column, an 800 mL column, a 900 mL column, a 1000 mL (1L) column, a 2000 mL (2L) column, a 10 L column, a 20 L column, a 30 L column, a 40 L column, a 50 L column, a 60 L column, a 70 L column, an 80 L column, a 90 L column, a 100L column, a 140 L column, or a column with a capacity greater than 140 L as well as any
  • the sample loading flow rate is less than or equal to the elution flow rate.
  • the loading flow rate may be in the range of about 10 mL/min to about 40 mL/min, about 15 mL/min to about 30 mL/min, or about 20 mL/min to about 25 mL/min, about 10 mL/min, about 20 mL/min, or about 30 cm/hr to about 135 cm/hr, for a 8 mL monolith column. Suitable flow rates may be extrapolated for a non-monolith column.
  • salt concentrations herein with reference to NaCl for convenience.
  • another salt of an equivalent ionic strength e.g., KC1
  • another salt having a different ionic strength e.g., but its concentration adjusted to an equivalent ionic strength (e.g., NH 4 AC), or a combination of salts, may be substituted.
  • a 1 : 1 electrolyte such as sodium chloride (NaCl), potassium chloride (KC1), formate (HCO2 ), or acetate (CH2CO2 ) (e.g., NH 4 Ac or NaAc)
  • NaCl sodium chloride
  • KC1 potassium chloride
  • HCO2 formate
  • CH2CO2 acetate
  • SO 4 2 sulfate
  • the ionic strength is four times higher.
  • one of skill in the art can substitute another salt, or a mixture of suitable salts, adjusted to the appropriate concentration to provide an ionic strength equivalent to that provided for NaCl.
  • salt equivalent e.g., “NaCl or equivalent”.
  • NaCl or equivalent a salt of NaCl with other salts, or a mixture of salts which do not include NaCl, but which are compatible with the apparatus and processes (e.g., affinity and/or anion exchange resin processes) described herein.
  • the novel FPLC strategy provided herein utilizes a strong anion exchange resin complex as described herein.
  • the anion exchange resin binds the rAAVl empty and full capsids are bound by a charge interaction while in buffer A (the running buffer).
  • buffer A the running buffer
  • the anion exchange resin column in equilibrated using Buffer A which contains about 200 nM NaCl to about 700 nM NaCl, or about 400 mM NaCl to about 650 mM NaCl, or salt equivalent.
  • Suitable buffers may include ions contributed from a variety of sources, such as, e.g., N-methylpiperazine;
  • Tris buffer component is selected.
  • Buffer A contains about 20 mM Tris-Cl, about 400 nM NaCl or equivalent, pH 7.5.
  • Buffer B is used to equilibrate the anion exchange resin.
  • Buffer B is preferably at a pH about 9.8 (preferably 9.8).
  • the buffer contains about 20 mM Bis-Tris Propane (BTP) and about 10 mM NaCl to about 40 nM NaCl (or salt equivalent).
  • a mixture containing rAAVl empty and full particles may be suspended in about 100% Buffer A and applied to the column (vessel).
  • the rAAVl particles and intermediates bind to the resin while other components are carried out in the buffer.
  • the total flow rate of the buffer is kept constant; however, the proportion of Buffer B (the “elution” buffer) is gradually increased from 0% to 100% according to a programmed change in concentration (the "gradient").
  • At least one nuclease digestion step is performed prior to loading the mixture onto the anion exchange resin, i.e. , during the harvest of the rAAV particles and intermediates from the production cell culture.
  • a second nuclease digestion step e.g., Benzonase
  • this may be performed during affinity capture.
  • an additional wash step may be incorporated into the affinity method in which the selected nuclease(s) are pre-mixed with a buffer and used in a wash step.
  • the buffer is at neutral pH and a relatively low salt concentration, e.g., about 10 to about 100 mM, about 20 mM to about 80 mM, about 30 mM NaCl to about 50 mL, or about 40 mM, based on the ionic strength of NaCl or a salt equivalent to any of the preceding ranges or amounts.
  • the flow rate for this wash step is performed at a slower rate than the other wash steps to allow for greater exposure of the nuclease to the loaded rAAV particles and intermediates.
  • the salt gradient has an ionic strength equivalent to at least about 10 mM NaCl to about 200 mM NaCl or salt equivalent. In another embodiment the salt gradient has an ionic strength equivalent to at least about 40 mM to about 190 mM NaCl, or about 70 nM to about 170 nM NaCl. In one embodiment, the AAV1 intermediates are separated from the anion exchange resin when the salt gradient reaches an ionic strength equivalent to about 50 nM NaCl or greater, or about 70 nM NaCl or greater.
  • the bound rAAVl particles and rAAVl empty intermediate dissociate and appear in the effluent.
  • the effluent passes through two detectors which measure salt concentration (by conductivity) and protein concentration (by absorption of ultraviolet light at a predetermined wavelength). However, other suitable detection means may be used.
  • salt concentration by conductivity
  • protein concentration by absorption of ultraviolet light at a predetermined wavelength.
  • other suitable detection means may be used.
  • the fractions under the 260 nm elution peak containing the rAAVl viral particles (“full") are collected and processed for further use.
  • the resulting rAAVl preparation or stock contains a ratio of particles to vector genomes of 1.
  • the rAAVl viral particles are placed in a suspension having a pH closer to a neutral pH which will be used for long-term storage and/or delivery to patients.
  • a pH may be in the range of about 6.5 to about 8, or about 7 to about 7.5.
  • the particles elute in a pH of about 9.8 and the rAAV particles are at least about 50- 90% purified from AAV1 intermediates, or a pH of 9.8 and about 90% to about 99% purified from AAV1 intermediates.
  • a stock or preparation of rAAVl particles (packaged genomes) is "substantially free" of AAV empty capsids when the rAAVl particles in the stock are at least about 75% to about 100%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least 99% of the rAAVl in the stock and "empty capsids" are less than about 1%, less than about 5%, less than about 10%, less than about 15% of the rAAVl in the stock or preparation.
  • the average yield of rAAV particles from loaded material is at least about 70%. This may be calculated by determining titer (genome copies) in the mixture loaded onto the column and the amount presence in the final elutions. Further, these may be determined based on q-PCR analysis and/or SDS- PAGE techniques such as those described herein (see figure legends) or those which have been described in the art.
  • Empty pt/mL divided by pt/mL and x 100 gives the percentage of empty particles.
  • methods for assaying for empty capsids and AAV vector particles with packaged genomes have been known in the art. See, e.g., Grimm et at., Gene Therapy (1999) 6: 1322-1330; Sommer et al, Molec. Ther. (2003) 7: 122-128.
  • the methods include subjecting the treated AAV stock to SD8- polyacryiamide gel electrophoresis, consisting of any gel capable of separating the three capsid proteins, for example, a gradient gel containing 3-8% Tris-acetate in the buffer, then running the gel until sample material is separated, and blotting the gel onto nylon or nitrocellulose membranes, preferably nylon.
  • Anti-AAV capsid antibodies are then used as the primary antibodies that bind to denatured capsid proteins, preferably an anti-AAV capsid monoclonal antibody, most preferably the Bl anti-AAV-2 monoclonal antibody (Wobus et al., J. Virol. (2000) 74:9281-9293).
  • a secondary antibody is then used, one that binds to the primary antibody and contains a means for detecting binding with the primary antibody, more preferably an anti-lgG antibody containing a detection molecule covalently bound to it, most preferably a sheep anti-mouse IgG antibody covalently linked to horseradish peroxidase.
  • a method for detecting binding is used to semi-quantitatively determine binding between the primary and secondary antibodies, preferably a detection method capable of detecting radioactive isotope emissions, electromagnetic radiation, or colorimetnc changes, most preferably a chemiluminescence detection kit.
  • samples from column fractions can be taken and heated in SDS-PAGE loading buffer containing reducing agent ⁇ e.g., DTT), and capsid proteins were resolved on pre-cast gradient polyacylamide gels (e.g. , Novex).
  • Silver staining may be performed using SilverXpress (Invitrogen, CA) according to the manufacturer's instructions, in one embodiment, the concentration of AAV vector genomes (vg) in column fractions can be measured by quantitative real time PCR (Q- PCR). Samples are diluted and digested with D ase 1 (or another suitable nuclease) to remove exogenous DNA.
  • the samples are further diluted and amplified using primers and a TaqManTM fluorogenic probe specific for the DNA sequence between the primers.
  • the number of cycles required to reach a defined level of fluorescence (threshold cycle, Ct) is measured for each sample on an Applied Biosystems Prsim 7700 Sequence Detection System.
  • Plasmid DNA containing identical sequences to that contained in the AAV vector is employed to generate a standard curve in the Q-PCR reaction.
  • the cycle threshold (Ct) values obtained from the samples are used to determine vector genome titer by normalizing it to the Ct value of the plasmid standard curve. End-point assays based on the digital PGR can also be used.
  • an optimized q-PCR method which utilizes a broad spectrum serine protease, e.g., proteinase K (such as is commercially available from Qiagen). More particularly, the optimized qPCR genome titer assay is similar to a standard assay, except that after the DNase I digestion, samples are diluted with proteinase K buffer and treated with proteinase K followed by heat inactivation. Suitably samples are diluted with proteinase K buffer in an amount equal to the sample size. The proteinase buffer may be concentrated to 2 fold or higher.
  • a broad spectrum serine protease e.g., proteinase K (such as is commercially available from Qiagen).
  • the optimized qPCR genome titer assay is similar to a standard assay, except that after the DNase I digestion, samples are diluted with proteinase K buffer and treated with proteinase K followed by heat inactivation. Suitably samples are diluted with proteinase K buffer in an amount equal
  • proteinase K treatment is about 0.2 mg/mL, but may be varied from 0.1 mg/mL to about 1 mg/mL.
  • the treatment step is generally conducted at about 55 °C for about 15 minutes, but may be performed at a lower temperature (e.g., about 37 °C to about 50 °C) over a longer time period (e.g., about 20 minutes to about 30 minutes), or a higher temperature (e.g., up to about 60 °C) for a shorter time period (e.g., about 5 to 10 minutes).
  • heat inactivation is generally at about 95 °C for about 15 minutes, but the temperature may be lowered (e.g., about 70 to about 90 °C) and the time extended (e.g., about 20 minutes to about 30 minutes). Samples are then diluted (e.g., 1000 fold) and subjected to TaqMan analysis as described in the standard assay.
  • droplet digital PGR may be used.
  • ddPCR droplet digital PGR
  • methods for determining single-stranded and self-complementary AAV vector genome titers by ddPCR have been described. See, e.g., M. Lock et al, Hu Gene Therapy Methods, Hum Gene Ther Methods. 2014 Apr;25(2): 115-25. doi: 10.1()89/hgtb.2()13.131. Epub 2014 Feb 14.
  • the mixture which is applied to the anion exchange resin has been purified from contamination with materials present from the production system.
  • the mixture comprising the recombinant AAVl viral particles and AAVl intermediates contains less than about 10% contamination from non- AAV viral and cellular proteinaceous and nucleic acid materials, or less than about 5% contaminants, or less than 1% contaminating viral and cellular proteinaceous and nucleic acid materials.
  • the mixture loaded onto the anion exchange resin is about 95% to about 99% free of contaminants.
  • contaminants refer to host cell, viral, and other proteinaceous materials which are present in the production culture or are by-products thereof. This term does not include rAAV particles or rAAV intermediates having formed AAV capsids.
  • a two-step chromatography method is used in which affinity capture is utilized to separate a mixture of recombinant AAVl viral particles and AAVl capsid intermediates from production system contaminants.
  • this processing has been found to allow approximately 3 times to 5 times the amount of starting material (based on the concentration of rAAV genome copies) to be processed using approximately 5 to 10 less resin, as compared to certain prior art approaches (e.g., one prior art approach utilized affinity capture after anion exchange and another utilized multiple, sequential, ion exchange resin columns).
  • the solid support is a cross-linked 6% agarose matrix having an average particle size of about 34 ⁇ and having an AAV-specific antibody.
  • affinity resin is AVB SepharoseTM high performance affinity resin using an AAV-specific camelid-derived single chain antibody fragment of llama origin which is commercially available from GE
  • AAV Sepharose The manufacturer's product literature indicates that the product binds AAVl, 2, 3 and 5, but has no reference to AAVl. The manufacturer's literature further recommends up to a 150cm/h flow rate and a relatively low loading salt concentration.
  • Other suitable affinity resins may be selected or designed which contain an AAV-specific antibody, AAVl specific antibody, or other immunoglobulin construct which is an AAV-specific ligand.
  • Such solid supports may be any suitable polymeric matrix material, e.g., agarose, sepharose, sephadex, amongst others.
  • Suitable loading amounts may be in the range of about 2 to about 5 x 10 15 GC, or less, based on the capacity of a 30-mL column. Equivalent amounts may be calculated for other sized columns or other vessels.
  • the term "genome copy" refers to the full particles in a mixture of both rAAVl full particles and rAAVl
  • the mixture is buffer exchanged with the column equilibration/loading buffer.
  • the method described herein utilizes a relatively high salt concentration for loading the column.
  • the mixture containing the AAVl viral particles and AAVl intermediates is loaded onto the affinity resin in a buffer having a high salt concentrations, e.g., about 400 nM NaCl to about 650 mM NaCl or another salt(s) having an equivalent ionic strength.
  • the wash step for the affinity resin is thereafter performed at an even higher salt concentration, e.g., in the range of about 750 mM to about 1 M NaCl or equivalent.
  • the AAVl mixture is maintained at a salt concentration of about 400 mM NaCl to about 650 mM NaCl, or equivalent prior to being applied to the anion exchange resin column.
  • the rAAVl mixture is maintained at this salt concentration following concentration and prior to loading onto the affinity resin.
  • a suitable buffer is AVB Buffer A, containing about 200 nM to about 600 nM NaCl, or about 400 nM NaCl, or the ionically equivalent of another salt, about 10 mM to about 40 mM Tris-Cl or another buffer, at a neutral pH.
  • the flow rate at loading may be a manufacturer's recommended value, e.g., about 149 cm/hr.
  • a wash step using AVB Buffer C is applied (1 M NaCl or an equivalent salt, 20 mM sodium citrate, neutral pH), followed by a wash with AVB Buffer A, and use of AVB Buffer B for elution.
  • AVB Buffer B is about 200 nM to about 600 nM NaCl, or about 400 nM NaCl, or the ionically equivalent of another salt, about 10 mM to about 40 mM Tris-Cl, or about 20 nM Tris-Cl or another buffer. In one embodiment, this step is performed at the range recommended by the
  • a low pH such as, e.g., about 2.5. In one embodiment, about 2 to about 8, or about 5 column volumes of buffer are used for these steps.
  • At least one nuclease digestion step is performed prior to loading the mixture onto the anion exchange resin, i.e. , during the harvest of the rAAV particles and intermediates from the production cell culture.
  • a second nuclease digestion step is performed during affinity capture.
  • an additional wash step may be incorporated into the affinity method in which the selected nuclease(s) are pre-mixed with a buffer and used in a wash step.
  • the buffer is at neutral pH and a relatively low salt concentration, e.g., about 20 to about 60 mM, about 30 mM NaCl to about 50 mL, or about 40 mM, based on the ionic strength of NaCl or a salt equivalent to any of the preceding ranges or amounts.
  • the flow rate for this wash step is performed at a slower rate than the other wash steps to allow for greater exposure of the nuclease to the loaded rAAV particles and intermediates.
  • a single nuclease, or a mixture of nucleases may be used in this step.
  • Such nucleases may target single stranded DNA, double-stranded DNA, or RNA.
  • DNase deoxyribonuclease
  • other suitable nucleases are known, many of which are commercially available.
  • a suitable nuclease or a combination of nucleases may be selected.
  • the nuclease(s) selected for this step may be the same or different from the nuclease(s) used during the processing preceding the affinity step and which more immediately follows harvest from the cell culture.
  • the load for the first affinity chromatography step is obtained following harvest and subsequent processing of cell lysates and/or supernatant of a production cell culture.
  • This processing may involve at least one of the following processes, including, optional lysis, optional collection from supernatant (media), filtrations, clarification, concentration, and buffer exchange.
  • rAAV production cultures for the production of rAAV virus particles may require; 1) suitable host cells, including, for example, human-derived cell lines such as HeLa, A549, or 293 cells, or insect-derived cell lines such as SF-9, in the case of baculovirus production systems; 2) suitable helper virus function, provided by wild type or mutant adenovirus (such as temperature sensitive adenovirus), herpes virus, baculovirus, or a nucleic acid construct providing helper functions in trans or in cis; 3) functional AAV rep genes, functional cap genes and gene products; 4) a transgene (such as a therapeutic transgene) flanked by AAV ITR sequences; and 5) suitable media and media components to support rAAV production.
  • suitable host cells including, for example, human-derived cell lines such as HeLa, A549, or 293 cells, or insect-derived cell lines such as SF-9, in the case of baculovirus production systems
  • suitable helper virus function provided by wild type
  • the cell itself may be selected from any biological organism, including prokaryotic (e.g. , bacterial) cells, and eukaryotic cells, including, insect cells, yeast cells and mammalian cells.
  • prokaryotic e.g. , bacterial
  • eukaryotic cells including, insect cells, yeast cells and mammalian cells.
  • Particularly desirable host cells are selected from among any mammalian species, including, without limitation, cells such as A549, WEHI, 3T3, 10T1/2, BHK, MDCK, COS 1, COS 7, BSC 1, BSC 40, BMT 10, VERO, WI38, HeLa, a HEK 293 cell (which express functional adenoviral El), Saos, C2C12, L cells, HT1080, HepG2 and primary fibroblast, hepatocyte and myoblast cells derived from mammals including human, monkey, mouse, rat, rabbit, and hamster.
  • the selection of the mammalian species providing the cells is not a limitation of this invention; nor is the type of mammalian cell, i.e. , fibroblast, hepatocyte, tumor cell, etc.
  • AAV sequences may be obtained from a variety of sources.
  • a suitable AAV sequence may be obtained as described in WO 2005/033321 or from known sources, e.g., the American Type Culture Collection, or a variety of academic vector core facilities.
  • suitable sequences are synthetically generated using known techniques with reference to published sequences. Examples of suitable AAV sequences are provided herein.
  • the cell contains the sequences which drive expression of an AAV capsid in the cell (cap sequences) and rep sequences of the same source as the source of the AAV ITRs found in the expression cassette, or a cross-complementing source.
  • the AAV cap and rep sequences may be independently selected from different AAV parental sequences and be introduced into the host cell in a suitable manner known to one in the art. While the full-length rep gene may be utilized, it has been found that smaller fragments thereof, i.e., the re/?78/68 and the rep52/40 are sufficient to permit replication and packaging of the AAV.
  • the host cell contains at least the minimum adenovirus DNA sequences necessary to express an Ela gene product, an Elb gene product, an E2a gene product, and/or an E4 ORF6 gene product.
  • the E2a gene product and/or E4 ORF6 gene product may be introduced via helper plasmid or by adenovirus co-infection.
  • the E2a gene product and/or E4 ORF6 may be substituted by herpes virus helper functions.
  • the host cell may contain other adenoviral genes such as VAI RNA, but these genes are not required.
  • the cell used does not carry any adenovirus gene other than El, E2a and/or E4 ORF6; does not contain any other virus gene which could result in homologous recombination of a contaminating virus during the production of rAAV; and it is capable of infection or transfection by DNA and expresses the transfected gene (s).
  • One cell type useful in the methods and systems described herein is a host cell stably transformed with the sequences encoding rep and cap, and which is transfected with the adenovirus El, E2a, and E40RF6 DNA and a construct carrying the expression cassette as described above.
  • Stable rep and/or cap expressing cell lines such as B-50 (International Patent Application Publication No. WO 99/15685), or those described in US Patent No. 5,658,785, may also be similarly employed.
  • Another desirable host cell contains the minimum adenoviral DNA which is sufficient to express E4 ORF6.
  • Yet other cell lines can be constructed using the novel modified cap sequences of the invention.
  • the preparation of a host cell involves techniques such as assembly of selected
  • DNA sequences may be accomplished utilizing conventional techniques. Such techniques include cDNA and genomic cloning, which are well known and are described in Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY., including polymerase chain reaction, synthetic methods, and any other suitable methods which provide the desired nucleotide sequence.
  • the required components for AAV production e.g. , adenovirus Ela, Elb, E2a, and/or E40RF6 gene products, rep or a fragment(s) thereof, cap, the expression cassette, as well as any other desired helper functions
  • one or more of the components required to be cultured in the host cell to package an expression cassette in an AAV capsid may be provided to the host cell in trans using a suitable genetic element.
  • Suitable media known in the art may be used for the production of rAAV vectors.
  • These media include, without limitation, media produced by Hy clone Laboratories and JRH including Modified Eagle Medium (MEM), Dulbecco's Modified Eagle Medium (DMEM), custom formulations such as those described in U.S. Pat. No. 6,566,118, and Sf-900 II SFM media as described in U.S. Pat. No. 6,723,551, each of which is incorporated herein by reference in its entirety, particularly with respect to custom media formulations for use in production of recombinant AAV vectors.
  • MEM Modified Eagle Medium
  • DMEM Dulbecco's Modified Eagle Medium
  • custom formulations such as those described in U.S. Pat. No. 6,566,118
  • Sf-900 II SFM media as described in U.S. Pat. No. 6,723,551, each of which is incorporated herein by reference in its entirety, particularly with respect to custom media formulations for use in production of recomb
  • rAAV production culture media may be supplemented with serum or serum- derived recombinant proteins at a level of 0.5%-20% (v/v or w/v).
  • rAAV vectors may be produced in serum-free conditions which may also be referred to as media with no animal-derived products.
  • commercial or custom media designed to support production of rAAV vectors may also be supplemented with one or more cell culture components know in the art, including without limitation glucose, vitamins, amino acids, and or growth factors, in order to increase the titer of rAAV in production cultures.
  • rAAV production cultures can be grown under a variety of conditions (over a wide temperature range, for varying lengths of time, and the like) suitable to the particular host cell being utilized.
  • rAAV production cultures include attachment-dependent cultures which can be cultured in suitable attachment- dependent vessels such as, for example, roller bottles, hollow fiber filters, microcarriers, and packed-bed or fluidized-bed bioreactors.
  • rAAV vector production cultures may also include suspension-adapted host cells such as HeLa, 293, and SF-9 cells which can be cultured in a variety of ways including, for example, spinner flasks, stirred tank bioreactors, and disposable systems such as the Wave bag system.
  • rAAV vector particles may be harvested from rAAV production cultures by lysis of the host cells of the production culture or by harvest of the spent media from the production culture, provided the cells are cultured under conditions known in the art to cause release of rAAV particles into the media from intact cells, as described more fully in US Patent No. 6,566,118).
  • Suitable methods of lysing cells include for example multiple freeze/thaw cycles, sonication, microfluidization, and treatment with chemicals, such as detergents and/or proteases.
  • rAAV production cultures may contain one or more of the following: (1) host cell proteins; (2) host cell DNA; (3) plasmid DNA; (4) helper virus; (5) helper virus proteins; (6) helper virus DNA; and (7) media components including, for example, serum proteins, amino acids, transferrins and other low molecular weight proteins.
  • the rAAV production culture harvest is clarified to remove host cell debris.
  • the production culture harvest is clarified by filtration through a series of depth filters including, for example, a grade DOHC Millipore Millistak+HC Pod Filter, a grade A1HC Millipore Millistak+ HC Pod Filter, and a 0.2 ⁇ Filter Opticap XL10 Millipore Express SHC Hydrophilic Membrane filter. Clarification can also be achieved by a variety of other standard techniques known in the art, such as, centrifugation or filtration through any cellulose acetate filter of 0.2 ⁇ or greater pore size known in the art. Still other suitable depth filters, e.g., in the range of about 0.045 ⁇ to about 0.2 ⁇ or other filtration techniques may be used.
  • the rAAV production culture harvest is treated with a nuclease, or a combination of nucleases, to digest any contaminating high molecular weight nucleic acid present in the production culture.
  • a DNAse e.g., Benzonase® digestion performed under standard conditions known in the art.
  • Benzonase® a final concentration of 1 unit/mL to 2.5 units/mL of Benzonase® is used at a temperature ranging from ambient temperature to 37° C for a period of 30 minutes to several hours, or about 2 hours.
  • a turbonuclease is used.
  • nuclease examples include other suitable nuclease, or a mixture of nucleases. Examples of other suitable nuclease is described earlier in this specification.
  • tangential flow filtration for concentrating the rAAV particles
  • heat inactivation of helper virus rAAV capture by hydrophobic interaction chromatography
  • buffer exchange by size exclusion chromatography SEC
  • nanofiltration may be used alone, in various combinations, or in different orders.
  • the method comprises all the steps in the order as described below.
  • the Benzonase®-treated mixture is concentrated via tangential flow filtration ("TFF").
  • TFF tangential flow filtration
  • Large scale concentration of viruses using TFF ultrafiltration has been described by R. Paul et al, Hu Gene Therapy, 4:609-615 (1993).
  • TFF concentration of the feedstream enables a technically manageable volume of feedstream to be subjected to the chromatography steps described herein and allows for more reasonable sizing of columns without the need for lengthy recirculation times.
  • the rAAV feedstream is concentrated between at least two-fold and at least ten-fold. In some embodiments, the feedstream is concentrated between at least ten-fold and at least twenty-fold.
  • the feedstream is concentrated between at least twenty -fold and at least fifty-fold.
  • TFF can also be used at any step in the purification process where it is desirable to exchange buffers before performing the next step in the purification process.
  • a virus particle includes one or more virus particles.
  • the terms “comprise”, “comprising”, “contain”, “containing”, and their variants are open claim language, i.e., are permissive of additional elements.
  • the terms “consists”, “consisting”, and its variants are closed claim language, i.e., exclusive additional elements.
  • references to "about” a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se.
  • description referring to "about X” includes description of "X.”
  • “about” refers to a variability of ⁇ 0.2 from the given value.
  • “about 9.8” encompasses to 9.6 to 10.0.
  • “about” refers to a variability of ⁇ 10% from a given value. In certain embodiments, the variability may be 1%, 5%, 10%, or values therebetween.
  • a scalable method for separating full AAVl viral particles from AAVl intermediates by using an anti-AAV antibody based affinity capture resin followed by an anion exchange resin is provided.
  • the mixture containing the AAVl viral particles and AAVl intermediates is loaded onto the affinity resin in a buffer having a high salt concentrations, e.g., about 400 nM NaCl to about 650 mM NaCl or another salt(s) having an equivalent ionic strength.
  • the wash step for the affinity resin is thereafter performed at an even higher salt concentration, e.g., in the range of about 750 mM to about 1 M NaCl or equivalent.
  • the AAVl mixture is maintained at a salt concentration of about 400 mM NaCl to about 650 mM NaCl, or equivalent prior to being applied to the anion exchange resin column.
  • the affinity capture includes a nuclease digestion step.
  • the rAAVl mixture is maintained at this salt concentration following concentration and prior to loading onto the affinity resin.
  • the affinity purified mixture containing the viral particles having packaged genomic sequences are separated from genome- deficient AAVl capsid intermediates by subjecting the mixture to fast performance liquid chromatography at a pH of about 10. More particularly, the AAVl viral particles and AAV1 intermediates are bound to an anion exchange resin equilibrated at a pH of about 9.8 and subjected to a salt gradient while monitoring eluate for ultraviolet absorbance at about 260 and about 280, wherein the AAV1 full capsids are collected from a fraction which is eluted when the ratio of A260/A280 reaches an inflection point.
  • a method for separating AAV1 viral particles from AAV1 capsid intermediates which involves:
  • Buffer 1% B which comprises a salt having the ionic strength of lOmM to 40 mM NaCl and BTP with a pH of about 9.8;
  • salt gradient is the equivalent of about about 10 mM to about 400 mM NaCl, or about 10 mM to about 200 mM, or about 10 mM to about 190 mM;
  • the intermediates are eluted from the anion exchange resin when the salt concentration is the equivalent of greater than about 50 mM NaCl.
  • Buffer A is further admixed with NaCl to a final concentration of 1M in order to form or prepare Buffer B.
  • the salt gradient has an ionic strength equivalent to 10 mM to about 190 mM NaCl.
  • the salt gradient has an ionic strength equivalent to 20 mM to about 190 mM NaCl, or about 20 mM to about 170 mM NaCl.
  • the elution gradient may be from 1% buffer B to about 19% Buffer B.
  • the vessel containing the anion exchange resin is a monolith column; loading, washing, and eluting occur in about 60 column volumes.
  • a method for separating recombinant AAVl viral particles containing DNA comprising genomic sequences from genome-deficient (empty) AAVl capsid intermediates involves:
  • the pH is 9.8 and the rAAV particles are at least about 90% purified from AAVl intermediates. In a further embodiment, the average yield of rAAV particles is at least about 70%.
  • the rAAVl producer cell culture is selected from a mammalian cell culture, a bacterial cell culture, and an insect cell culture, wherein said producer cells comprise at least (i) nucleic acid sequence encoding an AAVl capsid operably linked to sequences which direct expression of the AAVl capsid in the producer cells; (ii) a nucleic acid sequence comprising AAV inverted terminal repeat sequences and genomic transgene sequences for packaging into the AAV 1 capsid; and (iii) functional AAV rep sequences operably linked to sequences which direct expression thereof in the producer cells.
  • producer cells further comprise helper virus sequences required for packaging and replication of the AAV1 into a viral particle.
  • the material harvested from the cell culture is applied to an affinity resin to separate contaminants from AAV1 viral particles and empty AAV1 capsid intermediates.
  • the affinity resin separation comprises:
  • AAV vectors are produced by the triple transfection method in HEK293 cells as described previously (Lock et al. 2010, Hum Gene Ther, 21(1): 1259-1271).
  • a two-step chromatography purification scheme is described which selectively captures and isolates the genome-containing AAV vector particles from the clarified, concentrated supernatant of HEK 293 cells five days post transfection.
  • the load for the first chromatography step using AVB Sepharose High Performance affinity resin (GE) consists of the filter-clarified, 10X TFF-concentrated supernatant harvested from ten 36-layer Hyperstack cell culture vessels that have been previously treated at 37° C with 958,500 units of Benzonase for 2h followed by a hypertonic shock with 0.5 M NaCl for 2h.
  • the bulk harvest Prior to loading, the bulk harvest is buffer-exchanged with the column equilibration/loading buffer (AVB Buffer A) incubated overnight at 4 °C, and then filtered with a 0.2 ⁇ PES depth filter (Sartorius).
  • AVB Buffer A column equilibration/loading buffer
  • the sample is applied to a flow-packed, 30-ml AVB column with a bed height of approximately 15 cm using an AKTA Avant 150 liquid chromatography station (GE) and the following method:
  • a volume of 500 ⁇ of AVB Neutralization Buffer (0.01% Pluronic F-68, 0.2 M Bis-Tris propane, pH 9.8) is pre-added to the elution fraction tubes and upon completion of the run, the 5-ml fractions under the main 280-nm elution peak (typically three fractions) are pooled and diluted 50X with AEX Buffer A-9.8 (20 mM Bis-Tris Propane pH 9.8) plus Pluronic F-68 (0.001% final) in a polypropylene bottle.
  • Anion exchange chromatography is subsequently performed to separate the full or DNA-carrying viral particles from the contaminating empty particles in the second step.
  • the diluted column eluate from the capture step is applied to a pre-equilibrated CIMmultus QA-8ml monolith column (BIA Separations) and the following method is run:
  • AAV1 vector particles containing single stranded recombinant DNA genomes were produced by triple plasmid transfection in HEK293 cells (Lock et al. 2010, Hum Gene Ther, 21(1): 1259-1271) and the clarified production culture supernatant was purified by affinity chromatography on AVB resin (GE). The clarified supernatant was loaded to the affinity column at neutral pH in 400mM salt and eluted with a low pH (-2.5) buffer. The eluate was immediately adjusted back to neutral pH and then diluted 50-fold into a Bis-Tris-propane (BTP) buffer A at pH 9.5.
  • BTP Bis-Tris-propane
  • GC vector genome copies
  • a chromatogram of the CIMac -QA run is shown in Figure 1.
  • a major peak (PI) was observed in the elution gradient and the A260/A280 ratio of this peak was greater than one, as would be expected for a pure particle population containing vector genomes. Notably, 65% of the vector genomes loaded to the column were present in this peak as measured by the polymerase chain reaction.
  • a smaller peak (P2) was also observed in the gradient and in this case the peak absorbance at 280nm is higher than at 260nm.
  • a third peak (P3) was observed in the column strip and also had an A260/280 ratio of less than one.
  • a low A260/280 peak ratio ( ⁇ 1) is indicative of empty AAV particles lacking a vector genome.
  • the first peak (PI) is a full particle population and the last two peaks (P2 and P3) contain particle populations with low vector genome content.
  • P3 is retained on the column and only elutes in very high salt, while P2 elutes late in the elution gradient, suggests different surface charges for the two "empty" particle populations which likely results from different degrees of particle assembly and/or extents of genome packaging.
  • EXAMPLE 4 A separate AVB affinity resin-purified AAV1 vector preparation containing 2.64xl0 14 GC was loaded onto an 8mL CIMmultus-QA column in Bis-Tris-propane (BTP) buffer A at pH9.8 and at lOmL/min. The column was washed in buffer A with 20mM NaCl, eluted with a shallow (10-190mM NaCl, 60CV) salt gradient at 20mL/min and then stripped with high salt Buffer B (20mM BTP. pH9.8, 1M NaCl). Despite the increased amount of vector loaded, a chromatographic profile similar to that described previously (Example 3; Fig. 2) was obtained with a major peak (PI) in the elution gradient containing the majority of the vector genomes (Fig. 3).
  • BTP Bis-Tris-propane
  • the sample could be rerun at a higher pH and the salt gradient extended such that the front empty particle population and full particles are separated.
  • the results in this example demonstrate the scalability of the purification method and the robustness of full particle enrichment with scale.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Analytical Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • Virology (AREA)
  • Microbiology (AREA)
  • Immunology (AREA)
  • Biophysics (AREA)
  • Molecular Biology (AREA)
  • Plant Pathology (AREA)
  • Medicinal Chemistry (AREA)
  • Spectroscopy & Molecular Physics (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne un procédé de purification de chromatographie en deux étapes qui capture sélectivement et isole des particules de vecteurs de virus adéno-associé recombiné (rAAV) contenant le génome d'un surnageant concentré clarifié d'une culture de cellules de production rAAV. Le procédé utilise un procédé de capture par affinité réalisé à une concentration élevée en sel suivi d'un procédé de résine échangeuse d'anions réalisé à un pH élevé pour produire des particules de vecteurs rAAV sensiblement exemptes d'intermédiaires de rAAV.
PCT/US2016/065974 2015-12-11 2016-12-09 Procédé de purification évolutif pour virus adéno-associé (aav) 1 WO2017100674A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
US16/060,406 US11015173B2 (en) 2015-12-11 2016-12-09 Scalable purification method for AAV1
US17/328,687 US11713450B2 (en) 2015-12-11 2021-05-24 Scalable purification method for AAV1
US18/333,718 US20230323312A1 (en) 2015-12-11 2023-06-13 Scalable purification method for aav1

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US201562266351P 2015-12-11 2015-12-11
US62/266,351 2015-12-11
US201662322083P 2016-04-13 2016-04-13
US62/322,083 2016-04-13

Related Child Applications (2)

Application Number Title Priority Date Filing Date
US16/060,406 A-371-Of-International US11015173B2 (en) 2015-12-11 2016-12-09 Scalable purification method for AAV1
US17/328,687 Continuation US11713450B2 (en) 2015-12-11 2021-05-24 Scalable purification method for AAV1

Publications (1)

Publication Number Publication Date
WO2017100674A1 true WO2017100674A1 (fr) 2017-06-15

Family

ID=59013358

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2016/065974 WO2017100674A1 (fr) 2015-12-11 2016-12-09 Procédé de purification évolutif pour virus adéno-associé (aav) 1

Country Status (2)

Country Link
US (3) US11015173B2 (fr)
WO (1) WO2017100674A1 (fr)

Cited By (43)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10155931B2 (en) 2006-04-28 2018-12-18 The Trustees Of The University Of Pennsylvania Scalable production method for AAV
US10335466B2 (en) 2014-11-05 2019-07-02 Voyager Therapeutics, Inc. AADC polynucleotides for the treatment of parkinson's disease
WO2019173538A1 (fr) * 2018-03-06 2019-09-12 University Of Florida Research Foundation, Incorporated Chimères vaa
WO2019241535A3 (fr) * 2018-06-14 2020-01-23 Regenxbio Inc. Chromatographie d'échange d'anions pour la production de vaa recombinants
US10570395B2 (en) 2014-11-14 2020-02-25 Voyager Therapeutics, Inc. Modulatory polynucleotides
US10577627B2 (en) 2014-06-09 2020-03-03 Voyager Therapeutics, Inc. Chimeric capsids
US10584337B2 (en) 2016-05-18 2020-03-10 Voyager Therapeutics, Inc. Modulatory polynucleotides
US10597660B2 (en) 2014-11-14 2020-03-24 Voyager Therapeutics, Inc. Compositions and methods of treating amyotrophic lateral sclerosis (ALS)
WO2020172490A1 (fr) 2019-02-22 2020-08-27 The Trustees Of The University Of Pennsylvania Virus adéno-associé recombinant pour le traitement d'une neurodégénérescence d'apparition tardive chez l'adulte associée à grn
JP2020526190A (ja) * 2017-06-30 2020-08-31 スパーク セラピューティクス インコーポレイテッドＳｐａｒｋ Ｔｈｅｒａｐｅｕｔｉｃｓ， Ｉｎｃ． Ａａｖベクターのカラム精製方法
US10786568B2 (en) 2017-02-28 2020-09-29 The Trustees Of The University Of Pennsylvania AAV mediated influenza vaccines
US10792343B2 (en) 2013-03-15 2020-10-06 The Trustees Of The University Of Pennsylvania Compositions and methods for treating MPSI
US10973929B2 (en) 2016-02-03 2021-04-13 The Trustees Of The University Of Pennsylvania Gene therapy for treating mucopolysaccharidosis type I
US11098325B2 (en) 2017-06-30 2021-08-24 Intima Bioscience, Inc. Adeno-associated viral vectors for gene therapy
WO2022015715A1 (fr) 2020-07-13 2022-01-20 The Trustees Of The University Of Pennsylvania Compositions utiles pour le traitement de la maladie de charcot-marie-tooth
US11241506B2 (en) 2015-12-14 2022-02-08 The Trustees Of The University Of Pennsylvania Composition for treatment of Crigler-Najjar syndrome
WO2022046988A1 (fr) 2020-08-26 2022-03-03 The Trustees Of The University Of Pennsylvania Virus adéno-associé recombinant pour le traitement d'une neurodégénérescence d'apparition tardive chez l'adulte associée à grn
US11299751B2 (en) 2016-04-29 2022-04-12 Voyager Therapeutics, Inc. Compositions for the treatment of disease
US11298041B2 (en) 2016-08-30 2022-04-12 The Regents Of The University Of California Methods for biomedical targeting and delivery and devices and systems for practicing the same
US11326182B2 (en) 2016-04-29 2022-05-10 Voyager Therapeutics, Inc. Compositions for the treatment of disease
WO2022119890A1 (fr) 2020-12-01 2022-06-09 The Trustees Of The University Of Pennsylvania Compositions et leurs utilisations pour le traitement du syndrome d'angelman
EP3758724A4 (fr) * 2018-02-27 2022-07-06 The Trustees of The University of Pennsylvania Nouveaux vecteurs de virus adéno-associés (vaa), vecteurs de vaa présentant une désamidation de capside réduite et utilisations associées
WO2022165313A1 (fr) 2021-02-01 2022-08-04 Regenxbio Inc. Thérapie génique de céroïdes-lipofuscinoses neuronales
US11434502B2 (en) 2017-10-16 2022-09-06 Voyager Therapeutics, Inc. Treatment of amyotrophic lateral sclerosis (ALS)
WO2022221276A1 (fr) 2021-04-12 2022-10-20 The Trustees Of The University Of Pennsylvania Compositions utiles pour le traitement de l'amyotrophie spinale et bulbaire (sbma)
US11497576B2 (en) 2017-07-17 2022-11-15 Voyager Therapeutics, Inc. Trajectory array guide system
US11512327B2 (en) 2017-08-03 2022-11-29 Voyager Therapeutics, Inc. Compositions and methods for delivery of AAV
US11535665B2 (en) 2015-05-13 2022-12-27 The Trustees Of The University Of Pennsylvania AAV-mediated expression of anti-influenza antibodies and methods of use thereof
US11578341B2 (en) 2017-02-28 2023-02-14 The Trustees Of The University Of Pennsylvania Compositions useful in treatment of spinal muscular atrophy
US11603542B2 (en) 2017-05-05 2023-03-14 Voyager Therapeutics, Inc. Compositions and methods of treating amyotrophic lateral sclerosis (ALS)
WO2023056399A1 (fr) 2021-10-02 2023-04-06 The Trustees Of The University Of Pennsylvania Nouvelles capsides de vaa et compositions les contenant
US11697825B2 (en) 2014-12-12 2023-07-11 Voyager Therapeutics, Inc. Compositions and methods for the production of scAAV
WO2023133574A1 (fr) 2022-01-10 2023-07-13 The Trustees Of The University Of Pennsylvania Compositions et méthodes utiles pour le traitement de troubles médiés par c9orf72
US11752181B2 (en) 2017-05-05 2023-09-12 Voyager Therapeutics, Inc. Compositions and methods of treating Huntington's disease
US11759506B2 (en) 2017-06-15 2023-09-19 Voyager Therapeutics, Inc. AADC polynucleotides for the treatment of Parkinson's disease
WO2023196893A1 (fr) 2022-04-06 2023-10-12 The Trustees Of The University Of Pennsylvania Compositions et méthodes de traitement d'un cancer du sein métastatique her2 positif et d'autres cancers
US11827906B2 (en) 2017-02-28 2023-11-28 The Trustees Of The University Of Pennsylvania Adeno-associated virus (AAV) clade f vector and uses therefor
US11890329B2 (en) 2017-07-06 2024-02-06 The Trustees Of The University Of Pennsylvania AAV9-mediated gene therapy for treating mucopolysaccharidosis type I
WO2024036251A1 (fr) * 2022-08-12 2024-02-15 Ultragenyx Pharmaceutical Inc. Nouveaux procédés de chromatographie par échange d'anions pour la séparation de vide à partir de particules de virus adéno-associé recombinant pleines
US11931375B2 (en) 2017-10-16 2024-03-19 Voyager Therapeutics, Inc. Treatment of amyotrophic lateral sclerosis (ALS)
US11951121B2 (en) 2016-05-18 2024-04-09 Voyager Therapeutics, Inc. Compositions and methods for treating Huntington's disease
WO2024081551A1 (fr) * 2022-10-10 2024-04-18 Passage Bio, Inc. Procédé de purification de particules d'aav entièrement recombinés
US12023386B2 (en) 2021-12-28 2024-07-02 The Trustees Of The University Of Pennsylvania Composition for treatment of Crigler-Najjar syndrome

Families Citing this family (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017160360A2 (fr) 2015-12-11 2017-09-21 The Trustees Of The University Of Pennsylvania Méthode de purification évolutive pour virus adéno-associé 9 (aav9)
US11015173B2 (en) 2015-12-11 2021-05-25 The Trustees Of The University Of Pennsylvania Scalable purification method for AAV1
WO2017100704A1 (fr) 2015-12-11 2017-06-15 The Trustees Of The University Of Pennsylvania Procédé de purification évolutif d'aavrh10
EP4215605A1 (fr) 2015-12-11 2023-07-26 The Trustees of The University of Pennsylvania Procédé de purification évolutif d'aav8
MX2023005218A (es) 2020-11-03 2023-05-16 Pfizer Metodos de purificacion de vectores de aav mediante cromatografia de intercambio anionico.
US20220169991A1 (en) * 2020-12-01 2022-06-02 Global Life Sciences Solutions Usa Llc Viral vector purification apparatus and method
WO2024038365A1 (fr) 2022-08-16 2024-02-22 Pfizer Inc. Méthodes de purification de vecteurs de vaa par chromatographie d'échange d'anions
WO2024130070A2 (fr) 2022-12-17 2024-06-20 The Trustees Of The University Of Pennsylvania Capsides aav recombinantes avec motifs de ciblage spécifiques du muscle cardiaque et squelettique et leurs utilisations
WO2024130067A2 (fr) 2022-12-17 2024-06-20 The Trustees Of The University Of Pennsylvania Vecteurs mutants aav recombinants avec motifs de ciblage spécifiques des muscles cardiaque et squelettique et compositions les contenant

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0282177A2 (fr) * 1987-03-09 1988-09-14 Dionex Corporation Résine échangeuse d'ions comportant une couche extérieure de résine acrylique
US20080050770A1 (en) * 1998-12-01 2008-02-28 Introgen Therapeutics, Inc. Method for the production and purification of adenoviral vectors
US20090275107A1 (en) * 2006-04-28 2009-11-05 The Trustees Of The University Of Pennsylvania Scalable Production Method for AAV
US20130045186A1 (en) * 2001-11-13 2013-02-21 The Trustees Of The University Of Pennsylvania Method of Detecting and/or Identifying Adeno-Associated Virus (AAV) Sequences and Isolating Novel Sequences Identified Thereby
US20150024467A1 (en) * 2009-06-16 2015-01-22 Genzyme Corporation Methods for purification of recombinant aav vectors

Family Cites Families (43)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5658785A (en) 1994-06-06 1997-08-19 Children's Hospital, Inc. Adeno-associated virus materials and methods
EP0847442A1 (fr) * 1995-08-30 1998-06-17 Genzyme Corporation Purification d'adenovirus et de virus adeno-associe (aav) par voie chromatographique
CA2995542A1 (fr) 1997-09-05 1999-03-11 Genzyme Corporation Procedes de generation de preparations de vecteurs de aav recombinants dont le titre est eleve et qui sont exemptes de virus assistant
US6566118B1 (en) 1997-09-05 2003-05-20 Targeted Genetics Corporation Methods for generating high titer helper-free preparations of released recombinant AAV vectors
US6995006B2 (en) 1997-09-05 2006-02-07 Targeted Genetics Corporation Methods for generating high titer helper-free preparations of released recombinant AAV vectors
EP1015619A1 (fr) 1997-09-19 2000-07-05 The Trustees Of The University Of Pennsylvania Procedes et lignee cellulaire utiles pour la production de virus adeno-associes recombines
US6759237B1 (en) 1998-11-05 2004-07-06 The Trustees Of The University Of Pennsylvania Adeno-associated virus serotype 1 nucleic acid sequences, vectors and host cells containing same
AU768729B2 (en) 1998-11-05 2004-01-08 Trustees Of The University Of Pennsylvania, The Adeno-associated virus serotype 1 nucleic acid sequences, vectors and host cells containing same
AUPP702198A0 (en) 1998-11-09 1998-12-03 Silverbrook Research Pty Ltd Image creation method and apparatus (ART79)
US6893865B1 (en) 1999-04-28 2005-05-17 Targeted Genetics Corporation Methods, compositions, and cells for encapsidating recombinant vectors in AAV particles
US6593123B1 (en) 2000-08-07 2003-07-15 Avigen, Inc. Large-scale recombinant adeno-associated virus (rAAV) production and purification
US6723551B2 (en) 2001-11-09 2004-04-20 The United States Of America As Represented By The Department Of Health And Human Services Production of adeno-associated virus in insect cells
EP2359869B1 (fr) 2001-12-17 2018-12-26 The Trustees Of The University Of Pennsylvania Séquences de virus adéno-associés de sérotype 8 , vecteurs les contenant et leurs utilisations
ES2526341T3 (es) 2001-12-17 2015-01-09 The Trustees Of The University Of Pennsylvania Secuencias de serotipo 9 de virus adeno-asociado (AAV), vectores que las contienen, y usos de las mismas
US7419817B2 (en) 2002-05-17 2008-09-02 The United States Of America As Represented By The Secretary Department Of Health And Human Services, Nih. Scalable purification of AAV2, AAV4 or AAV5 using ion-exchange chromatography
SI2277996T1 (sl) 2003-05-21 2014-12-31 Genzyme Corporation Postopki za izdelavo pripravkov rekombinantnih AAV virionov v bistvu brez praznih kapsid
EP1486567A1 (fr) 2003-06-11 2004-12-15 Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts Vecteur derivé d'un virus associé aux adenovirus pour la thérapie génique
US9441244B2 (en) 2003-06-30 2016-09-13 The Regents Of The University Of California Mutant adeno-associated virus virions and methods of use thereof
EP2298926A1 (fr) 2003-09-30 2011-03-23 The Trustees of The University of Pennsylvania Clades (sous-types) et séquences de Virus Adeno-Associé (AAV), vecteurs les contenant et leurs utilistation
EP3409296A1 (fr) 2005-04-07 2018-12-05 The Trustees of the University of Pennsylvania Procédé d'amélioration de la fonction d'un vecteur aav
JP2007117003A (ja) 2005-10-28 2007-05-17 Nippon Pall Kk ウイルス中空粒子の迅速除去および精製方法
WO2007120542A2 (fr) 2006-03-30 2007-10-25 The Board Of Trustees Of The Leland Stanford Junior University Bibliothèque de capsides aav et protéines de capsides aav
US20090317417A1 (en) 2006-04-28 2009-12-24 The Trustees Of The University Of Pennsylvania Modified AAV Vectors Having Reduced Capsid Immunogenicity and Use Thereof
WO2009088786A1 (fr) 2007-12-31 2009-07-16 Nanocor Therapeutics, Inc. Interférence d'arn pour le traitement d'une insuffisance cardiaque
WO2009108274A2 (fr) 2008-02-26 2009-09-03 The University Of North Carolina At Chapel Hill Procédés et compositions pour virus adéno-associés (aav) présentant des mutations de la boucle hi
KR101010352B1 (ko) 2008-05-30 2011-01-25 삼성중공업 주식회사 전력 제어 장치 및 방법
EP2529020B1 (fr) 2010-01-28 2018-04-25 The Children's Hospital of Philadelphia Plateforme de fabrication évolutive pour purification de vecteurs viraux et vecteurs viraux ainsi purifiés utilisables en thérapie génique
CA3066596A1 (fr) 2010-04-23 2011-10-27 University Of Massachusetts Vecteurs aav ciblant le systeme nerveux central et leurs procedes d'utilisation
US8927514B2 (en) 2010-04-30 2015-01-06 City Of Hope Recombinant adeno-associated vectors for targeted treatment
PL2675902T3 (pl) 2011-02-17 2019-08-30 The Trustees Of The University Of Pennsylvania Kompozycje i sposoby do zmieniania specyficzności względem tkanki i poprawy transferu genu z udziałem aav9
JP6007463B2 (ja) 2011-04-18 2016-10-12 国立研究開発法人国立精神・神経医療研究センター 薬剤送達粒子及びその製造方法
US9169299B2 (en) 2011-08-24 2015-10-27 The Board Of Trustees Of The Leleand Stanford Junior University AAV capsid proteins for nucleic acid transfer
EP2954051B1 (fr) 2013-02-08 2019-03-27 The Trustees Of The University Of Pennsylvania Capsides aav8 modifiées pour transfert génétique pour thérapies rétiniennes
FR3002237B1 (fr) 2013-02-15 2017-12-15 Genethon Methodes pour la production de particules virales aav double brin
WO2014125647A1 (fr) 2013-02-18 2014-08-21 富士通オプティカルコンポーネンツ株式会社 Dispositif photorécepteur
ES2856090T3 (es) 2014-09-24 2021-09-27 Hope City Variantes de vectores de virus adenoasociados para la edición genómica de alta eficacia y sus métodos
EP3054006A1 (fr) * 2015-02-09 2016-08-10 Institut National De La Sante Et De La Recherche Medicale (Inserm) Purification de particule de virus adéno-associé recombinant avec une chromatographie à échange d'anions en plusieurs étapes
EP3054007A1 (fr) * 2015-02-09 2016-08-10 Institut National De La Sante Et De La Recherche Medicale (Inserm) Purification de particules de virus adéno-associés de recombinaison comprenant une étape de purification par affinité immunologique
MA48852A (fr) 2015-05-13 2020-04-01 Univ Pennsylvania Expression médiée par aav d'anticorps anti-grippaux et leurs procédés d'utilisation
EP4215605A1 (fr) 2015-12-11 2023-07-26 The Trustees of The University of Pennsylvania Procédé de purification évolutif d'aav8
US11015173B2 (en) 2015-12-11 2021-05-25 The Trustees Of The University Of Pennsylvania Scalable purification method for AAV1
WO2017160360A2 (fr) 2015-12-11 2017-09-21 The Trustees Of The University Of Pennsylvania Méthode de purification évolutive pour virus adéno-associé 9 (aav9)
WO2017100704A1 (fr) 2015-12-11 2017-06-15 The Trustees Of The University Of Pennsylvania Procédé de purification évolutif d'aavrh10

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0282177A2 (fr) * 1987-03-09 1988-09-14 Dionex Corporation Résine échangeuse d'ions comportant une couche extérieure de résine acrylique
US20080050770A1 (en) * 1998-12-01 2008-02-28 Introgen Therapeutics, Inc. Method for the production and purification of adenoviral vectors
US20130045186A1 (en) * 2001-11-13 2013-02-21 The Trustees Of The University Of Pennsylvania Method of Detecting and/or Identifying Adeno-Associated Virus (AAV) Sequences and Isolating Novel Sequences Identified Thereby
US20090275107A1 (en) * 2006-04-28 2009-11-05 The Trustees Of The University Of Pennsylvania Scalable Production Method for AAV
US20150024467A1 (en) * 2009-06-16 2015-01-22 Genzyme Corporation Methods for purification of recombinant aav vectors

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
LOCK ET AL.: "Analysis of Particle Content of Recombinant Adeno-Associated Virus Serotype 8 Vectors by Ion-Exchange Chromatography", HUMAN GENE THERAPY , PART B: METHOD, vol. 23, 4 January 2012 (2012-01-04), pages 56 - 64, XP055349796 *

Cited By (54)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11377641B2 (en) 2006-04-28 2022-07-05 The Trustees Of The University Of Pennsylvania Scalable production method for AAV
US10155931B2 (en) 2006-04-28 2018-12-18 The Trustees Of The University Of Pennsylvania Scalable production method for AAV
US10792343B2 (en) 2013-03-15 2020-10-06 The Trustees Of The University Of Pennsylvania Compositions and methods for treating MPSI
US10577627B2 (en) 2014-06-09 2020-03-03 Voyager Therapeutics, Inc. Chimeric capsids
US10335466B2 (en) 2014-11-05 2019-07-02 Voyager Therapeutics, Inc. AADC polynucleotides for the treatment of parkinson's disease
US11975056B2 (en) 2014-11-05 2024-05-07 Voyager Therapeutics, Inc. AADC polynucleotides for the treatment of Parkinson's disease
US11027000B2 (en) 2014-11-05 2021-06-08 Voyager Therapeutics, Inc. AADC polynucleotides for the treatment of Parkinson's disease
US10920227B2 (en) 2014-11-14 2021-02-16 Voyager Therapeutics, Inc. Compositions and methods of treating amyotrophic lateral sclerosis (ALS)
US10597660B2 (en) 2014-11-14 2020-03-24 Voyager Therapeutics, Inc. Compositions and methods of treating amyotrophic lateral sclerosis (ALS)
US11542506B2 (en) 2014-11-14 2023-01-03 Voyager Therapeutics, Inc. Compositions and methods of treating amyotrophic lateral sclerosis (ALS)
US10570395B2 (en) 2014-11-14 2020-02-25 Voyager Therapeutics, Inc. Modulatory polynucleotides
US11198873B2 (en) 2014-11-14 2021-12-14 Voyager Therapeutics, Inc. Modulatory polynucleotides
US11697825B2 (en) 2014-12-12 2023-07-11 Voyager Therapeutics, Inc. Compositions and methods for the production of scAAV
US11535665B2 (en) 2015-05-13 2022-12-27 The Trustees Of The University Of Pennsylvania AAV-mediated expression of anti-influenza antibodies and methods of use thereof
US11241506B2 (en) 2015-12-14 2022-02-08 The Trustees Of The University Of Pennsylvania Composition for treatment of Crigler-Najjar syndrome
US10973929B2 (en) 2016-02-03 2021-04-13 The Trustees Of The University Of Pennsylvania Gene therapy for treating mucopolysaccharidosis type I
US11326182B2 (en) 2016-04-29 2022-05-10 Voyager Therapeutics, Inc. Compositions for the treatment of disease
US11299751B2 (en) 2016-04-29 2022-04-12 Voyager Therapeutics, Inc. Compositions for the treatment of disease
US11951121B2 (en) 2016-05-18 2024-04-09 Voyager Therapeutics, Inc. Compositions and methods for treating Huntington's disease
US11193129B2 (en) 2016-05-18 2021-12-07 Voyager Therapeutics, Inc. Modulatory polynucleotides
US10584337B2 (en) 2016-05-18 2020-03-10 Voyager Therapeutics, Inc. Modulatory polynucleotides
US11298041B2 (en) 2016-08-30 2022-04-12 The Regents Of The University Of California Methods for biomedical targeting and delivery and devices and systems for practicing the same
US10786568B2 (en) 2017-02-28 2020-09-29 The Trustees Of The University Of Pennsylvania AAV mediated influenza vaccines
US11578341B2 (en) 2017-02-28 2023-02-14 The Trustees Of The University Of Pennsylvania Compositions useful in treatment of spinal muscular atrophy
US11827906B2 (en) 2017-02-28 2023-11-28 The Trustees Of The University Of Pennsylvania Adeno-associated virus (AAV) clade f vector and uses therefor
US11603542B2 (en) 2017-05-05 2023-03-14 Voyager Therapeutics, Inc. Compositions and methods of treating amyotrophic lateral sclerosis (ALS)
US11752181B2 (en) 2017-05-05 2023-09-12 Voyager Therapeutics, Inc. Compositions and methods of treating Huntington's disease
US11759506B2 (en) 2017-06-15 2023-09-19 Voyager Therapeutics, Inc. AADC polynucleotides for the treatment of Parkinson's disease
US11098325B2 (en) 2017-06-30 2021-08-24 Intima Bioscience, Inc. Adeno-associated viral vectors for gene therapy
JP2020526190A (ja) * 2017-06-30 2020-08-31 スパーク セラピューティクス インコーポレイテッドＳｐａｒｋ Ｔｈｅｒａｐｅｕｔｉｃｓ， Ｉｎｃ． Ａａｖベクターのカラム精製方法
EP3658250A4 (fr) * 2017-06-30 2021-10-27 Spark Therapeutics, Inc. Méthode de purification de colonne de vecteur aav
US11890329B2 (en) 2017-07-06 2024-02-06 The Trustees Of The University Of Pennsylvania AAV9-mediated gene therapy for treating mucopolysaccharidosis type I
US11497576B2 (en) 2017-07-17 2022-11-15 Voyager Therapeutics, Inc. Trajectory array guide system
US11512327B2 (en) 2017-08-03 2022-11-29 Voyager Therapeutics, Inc. Compositions and methods for delivery of AAV
US11434502B2 (en) 2017-10-16 2022-09-06 Voyager Therapeutics, Inc. Treatment of amyotrophic lateral sclerosis (ALS)
US11931375B2 (en) 2017-10-16 2024-03-19 Voyager Therapeutics, Inc. Treatment of amyotrophic lateral sclerosis (ALS)
EP3758724A4 (fr) * 2018-02-27 2022-07-06 The Trustees of The University of Pennsylvania Nouveaux vecteurs de virus adéno-associés (vaa), vecteurs de vaa présentant une désamidation de capside réduite et utilisations associées
US11905524B2 (en) 2018-03-06 2024-02-20 University Of Florida Research Foundation, Incorporated AAV chimeras
WO2019173538A1 (fr) * 2018-03-06 2019-09-12 University Of Florida Research Foundation, Incorporated Chimères vaa
JP7385603B2 (ja) 2018-06-14 2023-11-22 リジェネクスバイオ インコーポレイテッド 組換えａａｖ生成のためのアニオン交換クロマトグラフィー
WO2019241535A3 (fr) * 2018-06-14 2020-01-23 Regenxbio Inc. Chromatographie d'échange d'anions pour la production de vaa recombinants
JP2021526829A (ja) * 2018-06-14 2021-10-11 リジェネクスバイオ インコーポレイテッド 組換えａａｖ生成のためのアニオン交換クロマトグラフィー
WO2020172490A1 (fr) 2019-02-22 2020-08-27 The Trustees Of The University Of Pennsylvania Virus adéno-associé recombinant pour le traitement d'une neurodégénérescence d'apparition tardive chez l'adulte associée à grn
WO2022015715A1 (fr) 2020-07-13 2022-01-20 The Trustees Of The University Of Pennsylvania Compositions utiles pour le traitement de la maladie de charcot-marie-tooth
WO2022046988A1 (fr) 2020-08-26 2022-03-03 The Trustees Of The University Of Pennsylvania Virus adéno-associé recombinant pour le traitement d'une neurodégénérescence d'apparition tardive chez l'adulte associée à grn
WO2022119890A1 (fr) 2020-12-01 2022-06-09 The Trustees Of The University Of Pennsylvania Compositions et leurs utilisations pour le traitement du syndrome d'angelman
WO2022165313A1 (fr) 2021-02-01 2022-08-04 Regenxbio Inc. Thérapie génique de céroïdes-lipofuscinoses neuronales
WO2022221276A1 (fr) 2021-04-12 2022-10-20 The Trustees Of The University Of Pennsylvania Compositions utiles pour le traitement de l'amyotrophie spinale et bulbaire (sbma)
WO2023056399A1 (fr) 2021-10-02 2023-04-06 The Trustees Of The University Of Pennsylvania Nouvelles capsides de vaa et compositions les contenant
US12023386B2 (en) 2021-12-28 2024-07-02 The Trustees Of The University Of Pennsylvania Composition for treatment of Crigler-Najjar syndrome
WO2023133574A1 (fr) 2022-01-10 2023-07-13 The Trustees Of The University Of Pennsylvania Compositions et méthodes utiles pour le traitement de troubles médiés par c9orf72
WO2023196893A1 (fr) 2022-04-06 2023-10-12 The Trustees Of The University Of Pennsylvania Compositions et méthodes de traitement d'un cancer du sein métastatique her2 positif et d'autres cancers
WO2024036251A1 (fr) * 2022-08-12 2024-02-15 Ultragenyx Pharmaceutical Inc. Nouveaux procédés de chromatographie par échange d'anions pour la séparation de vide à partir de particules de virus adéno-associé recombinant pleines
WO2024081551A1 (fr) * 2022-10-10 2024-04-18 Passage Bio, Inc. Procédé de purification de particules d'aav entièrement recombinés

Also Published As

Publication number Publication date
US20210348132A1 (en) 2021-11-11
US20190002843A1 (en) 2019-01-03
US11713450B2 (en) 2023-08-01
US11015173B2 (en) 2021-05-25
US20230323312A1 (en) 2023-10-12

Similar Documents

Publication Publication Date Title
US20230323312A1 (en) Scalable purification method for aav1
US11732245B2 (en) Scalable purification method for AAV9
US11718835B2 (en) Scalable purification method for AAV8
US11713449B2 (en) Scalable purification method for AAVRH10
KR102669561B1 (ko) Aav 벡터 컬럼 정제 방법
EP4387750A1 (fr) Procédés de chromatographie par échange d'anions pour la purification de virus adéno-associés recombinants
RU2772876C2 (ru) Колоночные способы очистки вектора на основе aav
WO2022045055A1 (fr) PROCÉDÉ DE FORMULATION DE PARTICULES DE VECTEUR VIRTUEL NON ENVELOPPÉES PAR MODIFICATION DU pH
KR20240093848A (ko) Aav 벡터 컬럼 정제 방법
WO2024079655A1 (fr) Procédés de chromatographie pour la purification de capsides d'aav
WO2024081551A1 (fr) Procédé de purification de particules d'aav entièrement recombinés
CN117794629A (zh) Aav载体的柱纯化方法

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 16873988

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 16873988

Country of ref document: EP

Kind code of ref document: A1